The invention relates to a method for the preparation of a mixture of peptides having a cysteine- or cysteine/glycine content between 7-20 w/w %, to preparations comprising said peptides and to the use of such preparations as active compound in a medicament.
Peptides are herein defined as amino acid chains, derived from a protein; the molecular weight of the peptides is preferably between 200D and 8000D, more preferably between 1000D and 5000D.
In the art, there is a great demand for cysteine and cysteine/glycine comprising compounds for effective administration of said amino acids to the human or animal body. The availability of especially cysteine and to a lesser extent glycine, is a limiting factor in the syntheses of glutathion. Proper administration of cysteine, but also of glycine is therefore demanded in cases where an elevation of cellular glutathion levels in the human or animal body are needed.
Glutathion (GSH) is a tripeptide-thiol (L-xcex3-glutamyl-L-cysteinylglycine) having a broad range of vital functions, including protection of cells against oxygen intermediates, free radicals, by products of the oxygen requiring metabolism, and detoxification of xenobiotics. Further, glutathion seems to play a role in the prevention of cataract and oxidative DNA injury. Glutathion is therefore regarded as an important compound against oxidative stress related diseases like myocardial ischemia, cancer and cataract.
In view of the crucial role played by glutathion either in combatting the assaults of free radical injuries or in detoxification of xenobiotics, inclusing drug metabolites (such as cyclophosphamide, paraquat and acetaminophen) and in preventing peroxidation of cell components, a method for maintaining hepatic stores of glutathion, particularly during times of stress to the body, including chemotherapy, is needed.
In the art, various methods are known to increase cellular levels of glutathion. Administration to animals of the glutathion amino acid precursors glutamic acid, cysteine and glycine, may produce an increase in cellular glutathion, but there is a limit to the effectiveness of this procedure.
Cellular concentrations of GSH are dependent on the supply of cysteine, which is often the limiting amino acid, and which is derived from dietary protein and also by trans-sulfuration from methionine in the liver. However, administration of cysteine as free amino acid is not an ideal way to increase GSH concentrations because cysteine is rapidly metabolised and furthermore, appears to be toxic to cells at higher concentrations. Administration to animals of compounds that are transported into cells and converted intracellularly into cysteine is sometimes useful in increasing cellular glutathion levels.
Another way in which tissue GSH concentration may be increased is by administration of gamma glutamylcysteine or of gamma-glutamylcystine. The administered gamma-glutamyl amino acid is transported intact and serves as a substrate of GSH synthetase. It is also known that administration of N-acetyl-L-cysteine can often increase tissue concentrations of GSH. Other reports on using N-mercaptopropionyl glycine for increasing intracellular glutahion are known. A few clinical trials have been done using mercaptopropionyl glycine to elevate intracellular glutathion.
That the administration of glutathion itself might lead to increased glutathion levels has also been considered. However, there is no published evidence that shows that intact glutathion enters cells. In fact, there are several reports on particular biological systems indicating that glutathion itself is not transported into cells. The increase in cellular glutathion sometimes found after administration of glutathion is due to (a) extracellular breakdown of glutathion, (b) transport into cells of free amino acids or dipeptides derived from glutathion extracellularly, and (c) intracellular resynthesis of glutathion.
Apart from these conventional methods for increasing glutathion levels, there have been several attempts to demonstrate how glutathion can be enhanced intracellularly. All these relate to synthetic derivatives or about intact undenatured proteins which are heat labile and none whatsoever to natural derived peptide mixtures. Some of the relevant ones are summarised below:
U.S. Pat. No. 5,869,456 relates to preparation of pure alkyl esters of glutathion (95% pure) and a method for increasing intracellular glutathion levels by administering such alkyl diester of glutathion.
U.S. Pat. No. 5,464,825 describes the method for preparation and use of N-acyl glutathion monoalkyl esters to provide increased intracellular levels of glutathion or glutathion equivalents, e.g. N-acyl glutathion or glutathion monoalkyl esters.
U.S. Pat. No. 5,248,697 describes a method for maintaining and/or enhancing tissue or plasma levels of glutathion. The patent teaches the art of treatment of a mammal with a supranormal amount of glutamine, or a glutamine equivalent, to prevent the reduction in tissue glutathion levels associated with exposure of the mammal to a compound capable of oxidative injury to the tissue.
U.S. Pat. No. 4,665,082 discusses the role of L-2-oxothiazolidine-4-carboxylate, a sulfur analog of 5-oxoproline, cleaved by the enzyme-5-oxo-L-prolinase to form cysteine, thus providing the basis for a cysteine delivery system by the addition of L-2-oxothiazolidine-4-carboxylate to base amino acid solutions or by injecting it directly into in vivo cells.
DE patent No 4,329,857 teaches the use of thiol compounds (cysteine and its derivatives or analogues like N-acetyl cysteine, homocysteine, glutathion, 2-oxothiazolidine-4-carboxylic acid) as an agent for strengthening the immune system and immune reactions.
According to the present invention, a novel method for the preparation of a mixture of peptides having a cysteine content between 7-20 w/w % from a protein source, comprising cysteine containing proteins is provided. The protein source is preferably a natural protein source. The peptide mixture prepared according to this embodiment of the present invention has the advantage that it is derived from natural protein sources and will not show any adverse side-effects, whereas chemically produced cystein derivatives as mentioned in the prior art, have shown adverse side effects. There has been found that such a preparation of a peptide mixture can be very advantageously used as cysteine source in diet supplements or in medicaments, as will be explained below.
The method is characterized in that it comprises the steps of:
a) cleaving the proteins of the protein source into peptides;
b) digesting the peptides obtained in step a) by at least one exopeptidase, the action of which is at least attenuated at the position of a cysteine in the peptide, therewith forming digested peptides having a terminal cysteine;
c) purifying the digested peptides.
In the first step a) proteins of the protein source are cleaved into smaller peptides. This cleavage can be performed by cleavage reactions, known in the art; preferably, the cleavage is performed by enzymatic hydrolysis of the peptide bonds of the protein by e.g. an endopeptidase, resulting in the peptides of about the desired length, and therewith increasing the amount of substrate for the exopeptidase. In a second step, the peptides as obtained by the cleavage reaction, are digested by at least one exopeptidase. With xe2x80x9cat least one exopeptidasexe2x80x9d is meant that the digestion reaction can be carried out by one or more different exopeptidases. Exopeptidases release amino acids from the terminal ends of the peptides one by one. The exopeptidase and the digestion reaction conditions are chosen such, that the exopeptidase action is at least attenuated at the position of a cysteine in the peptide. With xe2x80x9cat least attenuatedxe2x80x9d is meant that the exopeptidase does not remove the cysteine from the peptide at the chosen reaction conditions or has very low preference for the cleavage of cysteine, therewith rendering said cleavage reaction very slow compared to cleavage of other amino acids from the peptide. By the use of such an exopeptidase and condition, the peptides are generated of which the terminal amino acids have been removed up to the cysteine residue most close to said terminus. The skilled person will be able to find conditions at which commercially available enzymes with exopeptidase function having attenuated action at the cysteine. It is to be understood that the peptides may have one or more amino acid chains that are coupled to each other by disulfide bridges of cysteine residues, present in the said amino acid chains. xe2x80x9cA digested peptide having a terminal cysteinexe2x80x9d therefore reflects to the fact that at least one of the termini of such a multi-chain peptide has a terminal cysteine. Of course, such a peptide may contain more than one terminal cysteine. Preferably, the enzymatic activity is inactivated before the purification step, e.g. by a pH shift or a thermal heat inactivation treatment.
Preferably, the exopeptidase comprises Carboxypeptidase Y (E.C.3.4.16.1.), as it has been found that this enzyme can be very effectively attenuated at cysteine residues, therewith producing peptides with terminal cysteine residues.
The cleavage step a) and the digestion step b) can be conducted simultaneously, e.g. by using an endopeptidase and an exopeptidase that both function at the same reaction conditions. Also, enzyme preparations can be used that have both endopeptidase and exopeptidase activity.
Finally, these digested peptides are purified. Suitable methods to purify the digested peptides from free amino acids, released by the exopeptidase, are known in the art. Since a difference in molecular weight is created between the cystine and glycine containing peptides and the other free amino acids, the cystine and glycine peptides can be purified using this difference. Several techniques, known in the art, could be used. Preferably the free amino acids are separated using a membrane process, preferably ultra or nanofiltration. The purification step can also advantageously comprise the use of an immobilized metal affinity chromatography step (IMAC) accordingly to Kronina et al., Journal of Chromatography A, 852 (1999) pp 261-272. The cysteine and glycine rich peptides can hereafter be dried.
In a special embodiment, the exopeptidase in step b) and the cleavage reaction are chosen such, that the exopeptidase is at least attenuated both at the position of a cysteine as well as of glycine in the peptide. This will result in digested peptides having predominantly a terminal cysteine or glycine.
The purified peptides, either enriched in cysteine residues or enriched in both cysteine and glycine residues, have shown to be very suitable sources for these limiting amino acids to be readily administered, in order to elevate the cysteine and glycine rates in the human or animal body, and may therefore elevate the intracellular glutathion levels.
The protein source may be any source as long as it comprises cysteine-containing proteins. In case a cysteine and glycine rich peptide preparation is to be produced, the protein source should contain proteins that contain glycine and cysteine.
Preferably, the protein source comprises at least two different proteins, that both contribute to the glycine and/or cysteine content of the peptides. One of the proteins may be glycine rich, whereas the second protein may be cystein rich. The protein source can also be prepared before being subjected to the method of the present invention, by e.g. two or more protein sources before or during the cleavage step.
Preferably, the protein source consists of edible proteins, so that the digested peptides can be used as food additive. In a very special embodiment, the protein source comprises whey protein isolates (WPI) and/or whey protein concentrates (WPC). The terms xe2x80x9cwhey protein isolatesxe2x80x9d and xe2x80x9cwhey protein concentratesxe2x80x9d are known in the field. Whey protein cincentrate is a whey protein product having 35-80 w/w % protein, whereas whey protein isolate has a protein content of 90 w/w % or higher. An example of WPC is Esprion 580 from DMV International; an example of WPI is Bipro from Bio-isolates Ltd. Whey protein is an important cysteine source and it is thought that whey protein concentrate induces glutathion production in animal organs, see e.g. U.S. Pat. No. 5,451,412. However, whey protein concentrates as such are not as suitable for the elevation of the intracellular glutathion levels compared to the peptides according to the present invention. The concentration of cystein and glycine in the intact whey proteins is much lower than in the peptides of the invention, and therefore requires much higher doses of the intact whey protein to reach an acceptable level of cystein in the application.
A further disadvantage of U.S. Pat. No. 5,451,412 is that the use of totally undenatured whey protein products can be very costly since it requires very delicate process conditions. Whey protein isolate comprises very suitable cysteine and glycine rich proteins, such as albumin, especially xcex1lactalbumin and bovine serum albumin. Said proteins are advantageously used in or as starting protein source of the method according to the invention.
In another preferred embodiment, the protein source comprises one or more of the group consisting of albumine, especially xcex1-lactalbumin, bovine serum albumin, egg proteins (e.g. ovalbumin, cystatin) wheat gluten, maize protein isolate.
Preferably, steps a) and b) are done at conditions, wherein sulfur bridges between cysteine residues as present in the proteins in the protein source are kept in the oxidised form as much as possible. In this way, cysteine-rich peptide mixtures are obtained, in which most of the cystein residues are oxidised and coupled to other peptides through disulfide bridges. Although the correct nomenclature for cysteins in oxidized form (i.e. being coupled to another cysteine residu by a sulfur bridge) is xe2x80x9ccystinexe2x80x9d, in this application xe2x80x9ccysteinexe2x80x9d is defined both as cysteine in the reduced form (having free SH-groups) as in the oxidized (cystine) form. Peptides, wherein the sulfur bridges between the cysteine residues are intact, may mimick parts of the native original protein from which the peptides are derived, therewith possibly conferring an improved biologic action compared to that of the separate peptides in reduced form. Further, the oxidized form is less reactive and therefor more stable in applications that undergo a heat treatment like pasteurization or sterilization.
A further advantage is the fact that many enzymes having exopeptidase activity do not cleave oxidized cysteines, whereas cysteines in reduced form may be cleaved by said enzymes from the peptides, although with a relative low activity. In order to produce peptide mixtures in native, i.e. undenatured form, steps a) and b) are preferably done at a pH between 2 and 8.
It is preferred to carry out the hydrolytic processes in acidic environments. At acid pH the disulfide bridges in cystine are more stable than at basic pH. [Creighton, T. E., 1993, Proteins: structures and Molecular Properties. 2nd Ed.; Freeman and Company, New York ]
It is preferred to cleave the proteins of the protein source in step a) by an enzyme with endopeptidase function. Using such an enzyme makes it possible to cleave the proteins under undenaturing (i.e. native) conditions, resulting in undenatured cleavage products. Physical or chemical cleavage mostly implicates application of denaturing conditions that can not be used if intact native peptide mixtures are to be obtained. For this, the expopeptidase digestion should also preferably take place at undenaturing conditions. The skilled person will know the proper conditions to yield intact native peptide mixtures. xe2x80x9cIntact native peptidexe2x80x9d is in this content to be understood as a peptide, having the same conformation as the said peptide has in the native, functional protein.
In a very attractive embodiment, the enzyme with endopeptidase function also has exopeptidase function, the exopeptidase function of which is attenuated at the position of cysteine or both at glycine and cysteine. Such enzymes are known in the art and the advantage thereof is that steps a) and b) can be done simultaneously. Examples of preferred enzymes having both endopeptidase as exopeptidase functions are Flavourzyme, Acid Protease A, Protease M, Protease 2A, Protease B, Corolase PN-L, Acid Protease or a combination of one or more thereof.
The invention further relates to preparations comprising cysteine-rich peptides, comprising 7-20 w/w % cysteine and to such a preparation comprising 7-20 w/w % of cysteine/glycine. As indicated above, said preparations can advantageously be used for administration to animals or humans in order to effectively improve the cysteine uptake of cysteine or a combination of cysteine and glycine for e.g. elevation of the intracellular glutathion level. Preferably, at least 80% of the peptides of the preparation comprises terminal cysteines and/or glycines, which are then readily available for the human or animal body. These terminal cysteines and/or glycines are obtained by the use of the exopeptidase as discussed above.
Further, the invention relates to the use of a preparation according to the invention as active compound in a medicament, especially in a medicament for treatment of conditions mediated by oxidative damage and in a medicament for the elevation of cellular glutathion levels in the human or animal body. For this, the preparation can be combined with any suitable carrier, diluent adjuvant etc. in order to obtain the medicament in the desired administration form. The preparation can also advantageously be used in an infant formula, e.g. in a breast milk substitute.
The invention is now illustrated in the following examples and figures which are meant to be illustrative only and not to limit the scope of the invention.